We study the mechanism and regulation of protein synthesis in eukaryotic cells focusing on regulation by GTP-binding proteins and protein phosphorylation. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is a GTP-binding protein and during the course of translation initiation the GTP is hydrolyzed to GDP. The eIF2 is released from the ribosome in complex with GDP and requires the guanine-nucleotide exchange factor eIF2B to convert eIF2-GDP to eIF2-GTP. This exchange reaction is regulated by a family of stress-responsive protein kinases that specifically phosphorylate the alpha subunit of eIF2 on serine at residue 51, and thereby covert eIF2 into an inhibitor of eIF2B. Among the family of eIF2alpha kinases are GCN2, which is activated under conditions of amino acid starvation, PKR, which is activated by double-stranded RNA and downregulates protein synthesis in virally infected cells, and PERK, activated under conditions of ER stress. Previously, in collaboration with Frank Sicheri, we determined the structure of the PKR kinase domain in complex with eIF2alpha. This structural analysis revealed that eIF2alpha binds to the C-terminal lobe making intimate contact with helix alphaG, while catalytic domain dimerization is mediated by a back-to-back orientation of the kinase N-terminal lobes. [unreadable] [unreadable] Consistent with the structural data, charge-reversal mutations that disrupt a conserved salt-bridge between Arg262 and Asp266 in the dimer interface block PKR autophosphorylation and eIF2alpha phosphorylation. Importantly, combining the two charge-reversal mutations in the same PKR allele, designed to restore the salt-bridge interaction with opposite polarity, rescued PKR activity. We proposed an ordered mechanism of PKR activation in which catalytic domain dimerization triggers autophosphorylation and specific substrate recognition. Interestingly, the residues forming the salt-bridge interaction in the PKR dimer interface are conserved among the eIF2alpha kinases. However, the crystal structure of the GCN2 kinase domain revealed a different dimerization interface than observed in the PKR structure. Whereas the PKR structure was of an active kinase, the GCN2 structure utilized a non-phosphorylated kinase domain and thus may represent an inactive kinase domain. The Arg262 and Asp266 residues that form an intermolecular salt-bridge in PKR are conserved in GCN2 and PERK; however, these two residues are too remote to interact in the GCN2 structure. To test the importance of this potential salt-bridge interaction in PKR, GCN2 and PERK, the residues constituting the salt-bridge were mutated either independently or together to residues with the opposite charge. Single mutations of the Asp (or Glu in PERK) and Arg residues blocked kinase function both in yeast cells and in vitro. However, for all three kinases the double mutation designed to restore the salt-bridge interaction with opposite polarity, resulted in a functional kinase (reference 1). Thus, the salt-bridge interaction and back-to-back dimer interface observed in the PKR structure is critical for the activity of all three eIF2alpha kinases. Our results are consistent with the notion that the PKR structure represents the active state of the eIF2alpha kinase domain while the GCN2 structure may represent an inactive state of the kinase.[unreadable] [unreadable] The GTP-binding (G) protein eIF5B catalyzes ribosomal subunit joining in the final step of translation initiation. The eIF5B is an ortholog of prokaryotic translation initiation factor IF2. Previous studies revealed that eIF5B consists of four domains that structurally assemble to form a chalice-shaped molecule. The G domain plus domains II and III form the cup of the chalice, a long alpha helix forms the stem, and domain IV is the base of the chalice. In addition, we previously showed that the domain IV of eIF5B binds to the C-terminal tail of the factor eIF1A (an ortholog of prokaryotic factor IF1). We have shown that the eIF5B-eIF1A interaction promotes ribosomal subunit joining and possibly provides a checkpoint for correct ribosome formation, with full activation of eIF5B GTP hydrolytic activity dependent on formation of a properly organized initiation complex. Whereas in vitro assays established that eIF5B catalyzes ribosomal subunit joining, supporting in vivo data has been lacking. To address the essential function of eIF5B in vivo, rapidly depleted eIF5B in yeast cells. Analysis of translation initiation complexes from these cells revealed a defect in subunit joining. Mutation of the eIF1A C-terminal tail impaired eIF5B binding to eIF1A, and overexpression of eIF5B suppressed the growth and translation initiation defects in yeast expressing the eIF1A mutant, indicating that eIF1A helps recruit eIF5B to the ribosome prior to subunit joining. Blocking GTP hydrolysis by eIF5B led to the accumulation of both eIF1A and eIF5B on the 80S products of subunit joining both in vivo and in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of a non-hydrolyzable GTP analog, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining, and that subsequent release of eIF1A is dependent on GTP hydrolysis and release of eIF5B from the 80S ribosome (reference 2).[unreadable] [unreadable] Our previous studies on an eIF5B Switch I mutant revealed that GTP hydrolysis by eIF5B activates a regulatory switch required for eIF5B release from the ribosome following subunit joining. To gain further insights into the eIF5B GTP-binding properties and regulatory switch, and by extension the switch and guanine-nucleotide binding behavior of other GTP-binding proteins, we conducted a mutational and suppressor analysis of the conserved Switch II Gly479 residue of yeast eIF5B. Based on studies of other G proteins, it has generally been thought that movement of this Gly residue is critical for the structural transition of Switch II during GTP binding and hydrolysis. We found that the G479A mutation in eIF5B impaired yeast cell growth and the guanine-nucleotide binding, GTP hydrolytic, and ribosomal subunit joining activities of eIF5B. In a screen for mutations that bypassed the critical requirement of this Switch II Gly in eIF5B, intragenic suppressors were identified in the Switch I element (A444V) and at a residue in domain 2 of eIF5B that interacts with Switch II (D740R). The intragenic suppressors restored yeast cell growth and eIF5B nucleotide binding, GTP hydrolysis, and subunit joining activities. We propose that the Switch II mutation distorts the geometry of the GTP-binding active site impairing nucleotide binding and the eIF5B domain movements associated with GTP binding. Accordingly, the Switch I and domain 2 suppressor mutations induce Switch II to adopt a conformation favorable for nucleotide binding and hydrolysis, and thereby re-establish coupling between GTP binding and eIF5B domain movements (reference 3). As the switch 2 mutation and the switch 1 suppressor mutation map to elements conserved in all GTP-binding proteins, we believe that this interaction may be of importance for all GTP-binding proteins.